A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Cancer
Cancer is the second largest cause of death worldwide. Cancer accounts for 13% of global mortality with more than 70% of cancer deaths occurring in low and middle-income countries where the prevalence of cancer is expected to increase as mortality from other diseases decreases. In the UK alone, a disease such as breast cancer kills over 12,000 women each year.
One approach to this problem has been to identify novel targets for cancer therapies and to use these to tailor the treatment of each patient according to the molecular make-up of their particular disease, rather than their overt clinical characteristics. While this has been in part successful, there are still a significant number of tumour types for which there are no targeted therapies and few treatment options other than surgery and cytotoxic chemotherapy.
PARP
There is now a significant body of evidence to suggest that inhibition of poly ADP ribose polymerase (PARP) superfamily proteins, such as PARP1, PARP2, Tankyrase 1 (also known as TNKS1, PARP5a) and Tankyrase 2 (also known as TNKS2, PARP5B) could have clinical utility. See, e.g., Krishnakumar et al., 2010. PARP superfamily members use beta-NAD+ as a substrate to generate ADP-ribose polymers on amino acid residues of protein acceptors. The result is a dramatic post-translational modification that can significantly alter the properties of the protein acceptor. See, e.g., Krishnakumar et al., 2010.
Although much of the focus has been on PARP1, studies over the past decade have identified a family of as many as 17 proteins that share homology to the catalytic domain of PARP1. In addition to the PARP-like domain, the PARP family members are “functionalized” with a wide variety of other structural and functional domains (e.g., DBDs, RNA-binding domains, subcellular localization signals, macrodomains, BRCT motifs, ankyrin repeats, zinc fingers) that determine their overall biological activities. Recently, a unified nomenclature referring to this family of proteins as ADP-ribosyl transferases (ARTs) has been proposed to recognize that fact that (1) PARPs catalyze a transferase reaction, not a template-dependent polymerization reaction; and (2) not all family members have PARP activity; some are likely to function as mono(ADP-ribosyl) transferases (mARTs). This new nomenclature is reflected in a recent structure-based classification of PARP family members into three groups based on their catalytic domains: (1) PARPs 1-5, which are bona fide PARPs containing a conserved glutamate (Glu 988 in PARP1) that defines the PARP catalytic activity; (2) PARPs 6-8, 10-12, and 14-16, which are confirmed or putative mARTs; and (3) PARPs 9 and 13, which lack key NAD-binding residues and the catalytic glutamate, and are likely inactive. See, e.g., Krishnakumar et al., 2010.
PARP family members localize to various cellular compartments, including the nucleus, cytoplasm, mitochondria, and vault particles, although the subcellular localization and function of many of the PARPs are unknown. The known functions of the PARP family members span a wide range of cellular processes, including DNA repair, transcription, cellular signalling, cell-cycle regulation, and mitosis. This diverse array of processes plays key roles in a wide variety of biological outcomes, including differentiation, development, stress responses, inflammation, and cancer. See, e.g., Krishnakumar et al., 2010.
The primary nuclear PARPs are PARP1, PARP2 (the closest paralog to PARP1), PARP3, and tankyrases 1 and 2. PARP1 is a very well studied protein and has a well-established role in DNA repair. See, e.g., Lord et al., 2008. Tankyrase 1 encompasses four distinct domains; the N terminal HPS domain (homopolymeric stretches of His, Pro and Ser); the ankyrin domain, containing 24 ANK repeats; a SAM (sterile alpha module) domain; and a C terminal PARP catalytic domain. See, e.g., Hsiao et al., 2008.
The best characterised function of tankyrase 1 is in telomere maintenance. The cellular machinery that normally replicates genomic DNA is unable to synthesise DNA at the telomere, the structure that caps the end of each chromosome. DNA synthesis at the telomere is instead carried out by telomerase. This enzyme complex consists of a RNA template and a DNA polymerase catalytic subunit. However, the activity of telomerase in most human somatic cells is relatively low and as such, attrition of the DNA at the telomere gradually occurs. This attrition of telomeric DNA is one of the factors that can lead to replicative senescence in somatic cells and this shortening of telomeres is often referred to as a “mitotic clock” that predetermines the replicative capacity of most cells. However, the situation in cancer cells is considerably different from that in somatic cells; up to 90% of all human cancer cells have a high level of telomerase activity. This increased level of telomere maintenance is one of the factors that enables tumour cells to avoid senescence and perpetually replicate. See, e.g., Harley, 2008.
The length of telomeric DNA is determined by a “protein counting” mechanism in which a series of telomere-bound proteins negatively regulate the access of telomerase to the telomere. For example, longer telomeres bind a larger number of DNA double strand-binding Telomeric Repeat Binding Factor (TRF1) proteins. Together with the TIN2-TPP1-POT1 protein complex, TRF1 blocks the access of telomerase to the 3′ DNA overhang at the end of chromosomes, thus limiting further extension of the telomere. Regulation of this process is controlled by tankyrase 1 which promotes telomeric extension by poly(ADP-ribosyl)ating TRF1, causing its release from the telomere and eventual proteasomal destruction. This release and degradation of TRF1 allows an enhanced level of telomerase access to the chromosome end and extension of the telomere. See, e.g., Harley, 2008.
Tankyrase 1 is also required after DNA replication in the S/G2 phase of the cell cycle to resolve sister chromatid cohesion before mitosis ensues. Depletion of tankyrase 1 in HeLa cells results in mitotic arrest. Persistent sister chromatid cohesion in tankyrase 1 depleted cells results in sister chromatid fusion. See, e.g., Hsiao et al., 2009. The mitotic defect in tankyrase-depleted cells may, in part, be determined by the tankyrase 1-mediated poly(ADP ribosyl)ation of the protein NuMA, which plays an essential role in organising microtubules at spindle pores. See, e.g., Chang et al., 2005.
Recent work has also suggested a role for Tankyrase 1 in the control of oncogenic Wnt signalling, most likely via a mechanism that involves the stabilisation of the Wnt signalling component, Axin. See, e.g., Huang et al., 2009. In this latter work and subsequent work (see, e.g., James et al., 2012; Bao et al., 2012; Casás-Selves et al., 2012; Waaler et al., 2012; Riffell et al., 2012) a number of investigators have shown that toolbox, non-drug like small molecule inhibitors of tankyrase can inhibit oncogenic Wnt signalling and can inhibit tumour cells that are addicted to Wnt signalling.
Wnt Signalling
Wnt signalling is an intracellular protein signalling network that transduces signals from cell surface bound receptors to a series of gene transcription events. In canonical Wnt signalling, Wnt ligands bind to cell-surface receptors of the Frizzled family; Frizzled bound receptors activate Dishevelled family proteins. In turn, activated Dishevelled proteins inhibit the function of a complex of proteins including Axin 1 and 2, GSK-3, and the protein APC. This Axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signalling molecule. When Wnt signalling is stimulated and Dishevelled proteins are active, the “β-catenin destruction complex” is inhibited, β-catenin degradation is reduced and β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors. This latter act drives a series of specific gene expression events that ultimately mediate Wnt signalling.
The association of dysregulated Wnt/β-catenin signalling with cancer has been well documented. Constitutively activated β-catenin signalling, caused either by APC deficiency or activating β-catenin mutations can lead to tumourigenesis. Furthermore, tankyrase is directly involved in the Wnt signalling cascade. Tankyrase PARylates both Axin 1 and Axin 2 and causes their degradation, driving β-catenin stabilisation/nuclear translocation and TCF/LEF mediated transcription. See, e.g., Huang et al., 2009. When tankyrase is inhibited, either genetically or with small molecules, Axin1 and 2 levels are stabilized and β-catenin degradation is enhanced, ultimately suppressing Wnt signalling, even in situations where Wnt signalling is usually constitutively elevated, such as APC deficiency. See, e.g., Huang et al., 2009. These data suggest that tankyrase inhibition could be used in order to modulate Wnt signalling, both in cancer, but also in other, non-cancer, pathologies where Wnt signalling is aberrant.
In addition to its effects on Wnt signally, it has also recently been demonstrated that silencing of tankyrase 1 by RNA interference is lethal in tumour cells with deficiencies in either of the breast cancer susceptibility proteins, BRCA1 and BRCA2, but not in wild type cells. BRCA mutation carriers with cancer still retain functional BRCA protein function in their normal cells, whilst it is lacking in tumour cells, suggesting that a tankyrase 1 inhibitor could be used to selectively target tumour cells in BRCA patients. See, e.g., McCabe et al., 2009b. This approach of combining tumour-specific genetic deficiencies with inhibition of a drug target to elicit a therapeutic window is an example of a “synthetic lethal” approach to the design of cancer therapies. See, e.g., Kaelin, 2009. This BRCA selective effect of tankyrase 1 inhibition may be caused by telomere attrition (caused by tankyrase 1 inhibition) and stalled replication forks (caused by BRCA deficiency) acting in concert to cause a threshold of DNA damage that is inconsistent with cell viability. Alternatively, synergistic defects in cytokinesis and sister chromatid segregation caused by BRCA deficiency and tankyrase 1 inhibition may also underlie the BRCA selective effect. See, e.g., Daniels, 2004. The use of tankyrase 1 inhibition in this context is described in McCabe et al., 2009a and McCabe et al., 2009b.
It has been shown that a proportion of patients without BRCA mutations have clinical characteristics, tumour morphologies and tumour molecular profiles that are reminiscent of BRCA mutation-associated cancer, a property termed BRCAness. See, e.g., Turner et al., 2004. This BRCAness phenotype is most well described in a significant number of patients with triple negative breast tumours. See, e.g., Turner et al., 2004. It has been shown that BRCA1 deficient, triple-negative breast cancer cell lines such as HCC1937 are particularly sensitive to tankyrase 1 inhibition. See, e.g., McCabe et al., 2009a and McCabe et al., 2009b. Inhibiting tankyrase 1 therefore, may be very effective in patients with germ-line BRCA mutations as well as patients whose tumours exhibit a BRCAness phenotype.
Non-Tumourigenic Mechanisms Modulated by Tankyrase
In addition to tankyrase inhibitors having potential as cancer therapeutics, a number of other studies suggest tankyrase inhibitors could be used in a number of other non-cancer related pathologies, the majority of which are driven by aberrant Wnt signalling, of which tankyrase activity is a rate limiting step (see, e.g., Riffell et al., 2012).
For example:
Recent work has indicated that inhibition of tankyrase can stabilize Axin2 levels in immature oligodendrocyte progenitor cells (OLPs) (see, e.g., Fancy et al., 2011). On the basis that Axin2 function is essential for normal kinetics of remyelination, tankyrase inhibition has been shown to accelerate OLP myelination after hypoxic and demyelinating injury (see, e.g., Fancy et al., 2011). This data suggest that small molecule tankyrase inhibitors might serve as pharmacological agents that could aid remyelination in neuropathies such as multiple sclerosis, neonatal hypoxic ischemic encephalopathy (HIE), and neonatal periventricular leukomalacia (PVL) (see, e.g., Fancy et al., 2011).
Other studies have also shown that tankyrase is essential for Herpes Simplex Virus replication (HSV). Efficient HSV-1 replication requires tankyrase PARP activity (see, e.g., Li et al., 2011). Further support for this hypothesis comes from the observation that HSV did not replicate efficiently in cells depleted of tankyrase 1. Moreover, tankyrase and the tankyrase substrate TRF2 (telomeric repeat binding factor 2) control the degradation of Ebstein-Barr Virus (EBV) DNA (see, e.g., Deng et al., 2002), suggesting tankyrase inhibitors could have utility as antiviral agents.
In addition, tankyrase inhibition is known to modulate glucose uptake (see, e.g., Yeh et al., 2007), suggesting that a small molecule tankyrase inhibitor could have utility in the treatment of metabolic diseases such as type 2 diabetes. In this case, tankyrase inhibition is thought to modulate glucose uptake by altering the function and cellular localisation of the glucose transporter type 4 (GLUT4) and the aminopeptidase IRAP (insulin-responsive aminopeptidase).
In addition, tankyrase inhibition is known to induce cardiomyocyte differentiation (see, e.g., Wang et al., 2011), suggesting that small molecule tankyrase inhibitors could have some ability in the treatment of cardiac disorders, such as cardiac repair after cardiac infarction.
In addition, tankyrase inhibition is know to minimise the pathological effects of lung fibrosis and tankyrase inhibitors can improve the survival of mice with bleomycin induced lung fibrosis (see, e.g., Distler et al., 2012) suggesting that small molecule tankyrase inhibitors could have some usefuleness in the treatment of lung disorders and fibrotic disorders such as pulmonary fibrosis, cystic fibrosis, cirrhosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's disease, keloid, scleroderma/systemic sclerosis and arthrofibrosis.
In addition to these pathologies, Wnt signalling and its modulation are also involved in a number of other pathogenic conditions suggesting that small molecules tankyrase inhibitors could have utility in these other Wnt related diseases, including:                Alzheimer's disease, where the Wnt mediator B-catenin activity is aberrant (see, e.g., Caricasole et al., 2003; Moon et al., 2004; Mudher and Lovestone, 2002);        Dupuytren skin disease, where the Wnt mediator B-catenin activity is also aberrant (see, e.g., Varallo et al., 2003);        tooth agenesis, where the Wnt mediator Axin2 activity is aberrant (see, e.g., Lammi et al., 2004);        osteoarthritis, where the Wnt mediator secreted frizzled-related protein 3 (FRP3) activity is aberrant (see, e.g., Loughlin et al., 2004);        exudative vitreoretinopathy, where the Wnt mediators frizzled family receptor 4 (FZD4) (see, e.g., Robitaille et al., 2002) and Norrie disease protein (see, e.g., Xu et al., 2004) activities are aberrant;        schizophrenia, where the Wnt mediators glycogen synthase kinase 3 beta (GSK3b) and wingless-type MMTV integration site family member 1 (Wnt1) are aberrant (see, e.g., Kozlovsky et al., 2002; Miyaoka et al., 1999);        osteoporosis, where the Wnt mediator low density lipoprotein receptor-related protein 5 (LRP5) activity is aberrant (see, e.g., Gong et al., 2001);        dermal hypoplasia, where the Wnt mediator porcupine homolog (PORCN) activity is aberrant (see, e.g., Grzeschik et al., 2007);        XX sex reversal, where the Wnt mediator R-spondin 1 (RSPO1) activity is aberrant (see, e.g., Parma et al., 2006);        anonychia and hyponychia, were the Wnt mediator R-spondin 4 (RSPO4) is aberrant (see, e.g., Bergmann et al., 2006; Blaydon et al., 2006);        sclerosteosis and Van Buchem disease, where the Wnt mediator sclerostin (SOST) activity is aberrant (see, e.g., Balemans et al., 2001; Balemans et al., 2002);        Fuhrmann syndrome, were the Wnt mediator wingless-related MMTV integration site 7A (Wnt7a) activity is aberrant (see, e.g., Woods et al., 2006);        Odonto-onchyo-dermal hypoplasia, where Wnt mediator wingless related MMTV integration site 10a (Wnt10a) activity is aberrant (see, e.g., Adaimy et al., 2007); and        early onset obesity, where the Wnt mediator wingless related MMTV integration site 10b (Wnt10b) activity is aberrant (see, e.g., Christodoulides et al., 2006).        
Moreover, aberrant telomerase protein component TERT expression and aberrant Wnt signalling are implicated in nephropathy, including HIV-associated nephropathy (see, e.g., Shkreli et al., 2011). Given the strong link between tankyrase inhibitors and modulation of both Wnt signalling and TERT function, it is likely that small molecule tankyrase inhibitors could be used in the treatment of these pathologies.
The inventors have identified a class of small molecule inhibitors of PARP superfamily members including PARP1 and Tankyrase 1 which are useful in the treatment of conditions, including proliferative conditions such as cancer. In some cases, these inhibitors are able to elicit biochemical inhibition of these targets as well as eliciting cellular activity including one or more or all of: (i) inhibition of Wnt signalling; (ii) inhibition of cell survival/proliferation; (iii) stabilisation of Axin and tankyrase levels; and (iv) formation of markers of DNA damage such as γH2AX foci.
It appears that the following 3-aryl-5-substituted-2H-isoquinolin-1-ones are known.
#StructureRegistry No.P01 70351-69-8 P02 70351-70-1 P03 70351-71-2 P04 70351-72-3 P05 203628-15-3 P06 203628-17-5 P07 203628-19-7 P08 220630-92-2 P09 223553-35-3 P10 884500-93-0 P11 884501-99-9 P121256940-02-9 P131256940-03-0 P141256940-06-3 P151256940-07-4 P161256940-08-5 P171256940-09-6 P181256940-10-9 P191256940-11-0 P201256940-12-1 P211256940-13-2 P221256940-16-5 P231256940-17-6 P241262335-24-9
It appears that the following 3-aryl-5-unsubstituted-2H-isoquinolin-1-ones are known.
#StructureRegistry No.P25 19069-81-9 P26 98659-53-1 P27 98659-55-3 P28 145104-33-2 P29 223552-86-1 P30 223553-20-6 P31 376354-94-8 P32 376354-97-1 P33 503613-43-2 P34 503613-44-3 P35 630423-61-9 P36 630423-64-2 P37 721960-58-3 P38 721960-60-7 P39 721960-73-2 P40 862469-72-5 P41 924299-93-4 P421044871-80-8 P431044871-83-1 P441193268-39-1 P451193268-40-4 P461253733-07-1 P471253733-10-6 P481417652-57-3